9. Pemeriksaan protein dan enzim
Protein dan Enzim
KIMIA KLINIK
Rochmy Istikharah
Clinical Significance
Clinical Significance
?
α-helix
β-sheet
Peptid
e bond
pH
Ionic strength
Temperature
Solvent (dielectric
constant)
Hormone receptors
Antibodies
Coenzymes
Denatura
tion
Electrophoresis
Isoelectric focusing
Ion-exchange chromatography
Clinical Significance
CONCENTRATION
♮ Total protein concentration
6-8 g/dL
♮ Selected protein conct.
PATTERN
Depend on
protein
propertie
s
Selected Protein
Analysis
Normal condition
8% albumin glycosylated
nonenzymatically
Hyperglycemia
(e.g. Diabetes Mellitus)
up to 25%
albumin glycosylated
nonenzymatically
Related with HbA1c in DM
Useful for assessing
diabetic control in
Px with hemolytic
Total Protein Analysis
Total Protein Analysis
–
OH
Cu2+ + Protein Cu2+-Protein complex
≥ 2 peptide bonds
Less sensitive than Lowry
!
Protein Analysis
•• Skrining
Skrining
•• Total
Total protein
protein 6-8
6-8 g/dL
g/dL dengan
dengan 3,5-5
3,5-5 g/dL
g/dL albumin
albumin
•• Pattern
Pattern of
of serum
serum protein
protein in
in agarose
agarose electrophoresis
electrophoresis
(Serum
(Serum protein
protein electrophoresis:
electrophoresis: SPE)
SPE)
Able to
diagnose
Unable to
diagnose
Selected
Protein
Analysis
Methods: Protein
Elektroforesis
Prinsip: Pemisahan protein berdasarkan perbedaan
muatan &/atau bobot molekul
(metode standar elektroforesis)
Protein Serum Elektroforesis
pI: isoelectric point (ampholyte & net charge = 0)
Horizontal electrophoresis
Cth. Agarose electrophoresis
Vertical electrophoresis
Cth. SDS-PAGE
Automated electrophoresis
Plasma Protein
Elektroforesis
SPE Patterns/Serum
PEP
Modified from
Vithoulkas H. HomeoNews
2008;9:9-10
SPE Patterns
Modified from
Vithoulkas H. HomeoNews
2008;9:9-10
SPE Patterns
Modified from
Vithoulkas H. HomeoNews
2008;9:9-10
IgA
SPE Patterns
Modified from
Vithoulkas H. HomeoNews
2008;9:9-10
Large spike in γ region
Homogenous Ig
immunofixation electrophoresis
O’Connel et al. Am Fam Physician
2005;71(1):105-112
McPherson & Pincus
Henry’s Clinical Diagnosis
21st Ed. 2006
Enzym
e
Kimia Klinik
Rochmy Istikharah
ENZIM
Glossary
•
Proenzim (Zymogen)
•
Enzim
•
Holoenzim
•
Apoenzim
•
Koenzim/kofaktor
•
Isoenzim
Plasma & Non-plasma
Specific Enzymes
Plasma
• Normal ada di plasma
Plasma adalah tempat
aksinya
Non-plasma
• Plasma bukan tempat
aksinya
• Konsentrasinya tinggi
dibanding organ
• Konsentrasinya di
plasma rendah (normal
krn sel apoptosis)
• Cth. Enzim pd
koagulasi
• Konsentrasi
meningkat???
• Apoptosis, nekrosis,
obat, etc
Harper’s
Biochemistry
26th Ed
• Regenerasi &
penyimpanan ATP
Kreatin + ATP
CK
Mg2+
Kreatin fosfat + ADP
• CK-BB: (CK-1)brain
type
• CK-MB: (CK2)hybrid type
• CK-MM: (CK3)Muscle type
Anderson, J. L. et al. J Am Coll Cardiol 2007;50:652-726
Copyright ©2007 American College of Cardiology Foundation. Restrictions may apply.
Faktor yg mempengaruhi
nilai rentang enzim
• Waktu sampling
• MI: CK-MB: 6 jam
• Usia
• Gender:
massa otot,
olahraga,
konsentrasi hormon
• Ras
• Olahraga
Alkaline phosphatase
(U/L)
[AMP buffer, p-nitrophenyl
phosphate substrate,
30oC]
18 th
40 – 100
CK serum
30 – 50 U/L
500 - 1000 U/L
Pengukuran Konsentrasi
Enzim
Pengukuran Tidak
Langsung:
aktivitas enzim
1.Single reaction
Pengukuran produk
atau substrat yg
dikatalisis oleh
enzim tsb jarang
2.Coupled reaction
Pemeriksaan
Langsung:
Immunoassay
• Monoclonal/polyc
lonal antibodies
• Mengukur
konsentrasi enzim itu
sendiri
• Contoh teknik: ELISA
• Suhu
• Suhu dijaga dlm rentang ±0,1o
• Perbedaan 10o aktivitas enzim meningkat 2xnya
• pH: pH optimum utk enzim aktif
• Garam
• Terlalu tinggi aktivitas enzim turun drastis
• Konsentrasi protein:
• Plasma manusia, protein: 70 g/L; Urin hampir tidak ada
• Penambahan albumin pd pemeriksaan amilase di urin
(spike)
• Inhibitor & pengganggu lain
KIMIA KLINIK
Rochmy Istikharah
Clinical Significance
Clinical Significance
?
α-helix
β-sheet
Peptid
e bond
pH
Ionic strength
Temperature
Solvent (dielectric
constant)
Hormone receptors
Antibodies
Coenzymes
Denatura
tion
Electrophoresis
Isoelectric focusing
Ion-exchange chromatography
Clinical Significance
CONCENTRATION
♮ Total protein concentration
6-8 g/dL
♮ Selected protein conct.
PATTERN
Depend on
protein
propertie
s
Selected Protein
Analysis
Normal condition
8% albumin glycosylated
nonenzymatically
Hyperglycemia
(e.g. Diabetes Mellitus)
up to 25%
albumin glycosylated
nonenzymatically
Related with HbA1c in DM
Useful for assessing
diabetic control in
Px with hemolytic
Total Protein Analysis
Total Protein Analysis
–
OH
Cu2+ + Protein Cu2+-Protein complex
≥ 2 peptide bonds
Less sensitive than Lowry
!
Protein Analysis
•• Skrining
Skrining
•• Total
Total protein
protein 6-8
6-8 g/dL
g/dL dengan
dengan 3,5-5
3,5-5 g/dL
g/dL albumin
albumin
•• Pattern
Pattern of
of serum
serum protein
protein in
in agarose
agarose electrophoresis
electrophoresis
(Serum
(Serum protein
protein electrophoresis:
electrophoresis: SPE)
SPE)
Able to
diagnose
Unable to
diagnose
Selected
Protein
Analysis
Methods: Protein
Elektroforesis
Prinsip: Pemisahan protein berdasarkan perbedaan
muatan &/atau bobot molekul
(metode standar elektroforesis)
Protein Serum Elektroforesis
pI: isoelectric point (ampholyte & net charge = 0)
Horizontal electrophoresis
Cth. Agarose electrophoresis
Vertical electrophoresis
Cth. SDS-PAGE
Automated electrophoresis
Plasma Protein
Elektroforesis
SPE Patterns/Serum
PEP
Modified from
Vithoulkas H. HomeoNews
2008;9:9-10
SPE Patterns
Modified from
Vithoulkas H. HomeoNews
2008;9:9-10
SPE Patterns
Modified from
Vithoulkas H. HomeoNews
2008;9:9-10
IgA
SPE Patterns
Modified from
Vithoulkas H. HomeoNews
2008;9:9-10
Large spike in γ region
Homogenous Ig
immunofixation electrophoresis
O’Connel et al. Am Fam Physician
2005;71(1):105-112
McPherson & Pincus
Henry’s Clinical Diagnosis
21st Ed. 2006
Enzym
e
Kimia Klinik
Rochmy Istikharah
ENZIM
Glossary
•
Proenzim (Zymogen)
•
Enzim
•
Holoenzim
•
Apoenzim
•
Koenzim/kofaktor
•
Isoenzim
Plasma & Non-plasma
Specific Enzymes
Plasma
• Normal ada di plasma
Plasma adalah tempat
aksinya
Non-plasma
• Plasma bukan tempat
aksinya
• Konsentrasinya tinggi
dibanding organ
• Konsentrasinya di
plasma rendah (normal
krn sel apoptosis)
• Cth. Enzim pd
koagulasi
• Konsentrasi
meningkat???
• Apoptosis, nekrosis,
obat, etc
Harper’s
Biochemistry
26th Ed
• Regenerasi &
penyimpanan ATP
Kreatin + ATP
CK
Mg2+
Kreatin fosfat + ADP
• CK-BB: (CK-1)brain
type
• CK-MB: (CK2)hybrid type
• CK-MM: (CK3)Muscle type
Anderson, J. L. et al. J Am Coll Cardiol 2007;50:652-726
Copyright ©2007 American College of Cardiology Foundation. Restrictions may apply.
Faktor yg mempengaruhi
nilai rentang enzim
• Waktu sampling
• MI: CK-MB: 6 jam
• Usia
• Gender:
massa otot,
olahraga,
konsentrasi hormon
• Ras
• Olahraga
Alkaline phosphatase
(U/L)
[AMP buffer, p-nitrophenyl
phosphate substrate,
30oC]
18 th
40 – 100
CK serum
30 – 50 U/L
500 - 1000 U/L
Pengukuran Konsentrasi
Enzim
Pengukuran Tidak
Langsung:
aktivitas enzim
1.Single reaction
Pengukuran produk
atau substrat yg
dikatalisis oleh
enzim tsb jarang
2.Coupled reaction
Pemeriksaan
Langsung:
Immunoassay
• Monoclonal/polyc
lonal antibodies
• Mengukur
konsentrasi enzim itu
sendiri
• Contoh teknik: ELISA
• Suhu
• Suhu dijaga dlm rentang ±0,1o
• Perbedaan 10o aktivitas enzim meningkat 2xnya
• pH: pH optimum utk enzim aktif
• Garam
• Terlalu tinggi aktivitas enzim turun drastis
• Konsentrasi protein:
• Plasma manusia, protein: 70 g/L; Urin hampir tidak ada
• Penambahan albumin pd pemeriksaan amilase di urin
(spike)
• Inhibitor & pengganggu lain